1. Field of the Invention
This invention relates to ribozymes that cleave RNA. More specifically it reflects the enhancement of ribozyme catalytic activity by modifying ribozyme flanking sequence nucleotides to have substituents on the oxygen at the 2xe2x80x2-position, and by using a facilitator oligonucleotide complementary to an RNA sequence contiguous to the ribozyme.
2. Description of the Related Art
Drugs might be based on RNA catalysts or enzymes (ribozymes) designed to cleave viral or messenger RNA with high specificity at a rapid rate. These requirements historically have been mutually limiting.
Ribozymes consist of a catalytic core having flanking sequences adjacent the core which hybridize to the substrate RNA. The simplest ribozyme is an RNA motif known as a hammerhead.
Among the factors which limit ribozyme activity in cells are the extent of cellular uptake of the ribozyme and the extent of ribozyme degradation by nucleases. Accordingly, ribozymes having increased resistance to nuclease degradation are desired.
Ribozyme specificity depends on the number of base pairs formed between the ribozyme flanking sequences and its RNA substrate. Increased base pairing has been shown to decrease the rate of cleavage. Goodchild and Kohli, Arch. Biochem. Biophys., 284: 386-391 (1991). Goodchild and Kohli studied the cleavage of a sequence from HIV-1 RNA by various hammerhead ribozymes and determined that the rate of cleavage was dependent on the length of the flanking sequence. Shorter sequences were shown to result in weaker binding between the ribozyme and the cleavage. products together with increased rate of cleavage. A ribozyme with 12 bases in the flanking sequences cleaved 10 times faster then one with 20 bases.
However, to have the requisite selectivity or specifity, i.e., the ability to discriminate between all RNA molecules in a cell, a ribozyme must form a minimum of about 15 base pairs with the target substrate. This requirement for selectivity limits the rate of cleavage that may be realized.
Accordingly, ribozymes having increased catalytic activity or methods of increasing ribozyme catalytic activity are needed.
Uhlenbeck, Nature, 328: 596-600 (1987) describes the synthesis of two oligoribonucleotides that can combine to form a structure consistent with the consensus self-cleaving domain. Because rapid cleavage of one of the oligomers was observed only when the other was present, the domain was necessary and sufficient for cleavage. The properties of the cleavage reaction were studied in detail. Nearly complete cleavage occurred even with large excess of the oligomer that was cleaved. This indicates that the oligomer that is uncleaved can cycle in the reaction and therefore be considered to act as a catalyst in the cleavage of the other oligomer.
Haseloff and Gerlach, Nature 334: 585-59 (1988), discuss the dissection of the RNA substrate and enzyme activities from a single self-cleaving domain from the (+) strand of the satellite RNA of tobacco ringspot virus (sTobRV). Inspection of the separated substrate and ribozyme activities, in comparison with other naturally-occurring self-cleaving domains, led to a model for the design of oligoribonucleotides which posses new and highly sequence-specific endoribonuclease activities. This model was successfully tested by the design and construction of ribozymes targeted against three sites within the Tn9 chloramphenicol acetyl-transferase (CAT) messenger RNA sequence.
Chemical modifications to nucleotides in the central region of various hammerhead ribozymes have been attempted; no such modifications have resulted in increased catalytic activity. In fact, almost all such reported modifications have resulted in decreased catalytic activity for the ribozymes.
Perreault, et al., Nature 344: 565-567 (1990) reports the results of replacing ribonucleotides in a ribozyme with deoxyribonucleotides. Analysis of the cleavage products of several of the hammerhead analogs indicated the involvement and the reaction of the 2xe2x80x2-OH adjacent to the cleavage site in a substrate. This analysis demonstrated that some 2xe2x80x2-OH groups in the catalytic region affect activity. The introduction of 2xe2x80x2-deoxynucleotides at the conserved positions E 13, 14 and 27-29 within the ribozyme sequence resulted in a 96% decrease of catalytic efficiency.
In addition, Perreault et al., Biochemistry 30: 4020-4025 (1991), and Dahn and Uhlenbeck, Biochemistry 72: 819-23 (1990) report that the replacement of various 2xe2x80x2-hydroxyl groups with hydrogen atoms reduced the catalytic activity of hammerhead ribozymes.
Olsen et al., Biochemistry 30: 9735-9741 (1991), report that replacing 2xe2x80x2-hydroxyl groups on all adenosine residues by either fluorine or hydrogen produced a large decrease in catalytic activity.
Pieken et al., Science 253: 314-317 (1991), report that catalytic activity was reduced by replacing various 2xe2x80x2-hydroxyl groups on adenosine residues by fluorine and by replacing the 2xe2x80x2-hydroxyl groups on cytidine residues by amine groups. However, catalytic activity was unaltered by replacing the 2xe2x80x2-hydroxyl groups on cytidine residues by fluorine or the 2xe2x80x2-hydroxyl groups on uridine residues by fluorine or amino groups.
Odai et al., FEBS Letters 267: 150-152 (1990), report that replacing by hydrogen the exocyclic amino group of a conserved guanosine residue in the core region reduced catalytic activity.
Ruffner and Uhlenbeck, Nucleic Acids Research 18: 6025-6029 (1990), and Buzayan et al., Nucleic Acids Research 18: 4447-4451 (1990), disclose that replacing oxygen atoms by sulfur on various internucleotide phosphate residues reduced catalytic activity.
Fedor and Uhlenbeck, Proc. Natl. Acad. Sci. USA 87: 1668-1672 (1990), analyzed the kinetics of cleavage for several hammerhead sequences to characterize the reaction mechanism and explore how nucleotides involved in substrate binding affect cleavage.
Goodchild et al., Arch. Biochem. Biophys. 263: 401-409 (1988) discusses the effects of a series of synthetic oligonucleotides (hybridons) complementary to the 5xe2x80x2 non-coding regions of rabbit xcex2-globin mRNA on endogenous protein synthesis in a rabbit reticulocyte cell-free translation system. With highly purified hybridons inhibition was completely specific for beta globin. Mixtures of two oligonucleotides binding contiguously to the mRNA were more effective than either oligomer alone.
Maher and Dolnick, Nucleic Acids Res. 16: 3341-3358 (1988) report that antisense oligonucleotides containing either anionic diester or neutral methylphosphonate internucleoside linkages were prepared by automated synthesis, and subsequently compared for their ability to arrest translation of human dihydrofolate reductase (DHFR) mRNA in a nuclease treated rabbit reticulocyte lysate. In the case of oligodeoxyribonucleotides, tandem targeting of three 14-mers resulted in synergistic and complete selective inhibition of DHFR synthesis at a total oligomer concentration of 25 xcexcM.
Kutyavin et al, FEBS Lett. 238: 35-38 (1988) report that mono- and diphenazinium derivatives of oligonucleotides complementary to the DNA sequence adjacent to the target sequence of the addressed alkylation of DNA significantly enhance the extent and specificity of alkylation by p-(N-2-chloroethyl-N-methylamino(benzylamido) derivatives of the addressing oligonucleotides.
Inoue et al., Nucleic Acids Res. 15: 6131-6148 (1977) determined that (1) a 2xe2x80x2-O-methyl oligodeoxyribonucleotide-RNA duplex was much more stable than the corresponding oligodeoxy ribonucleotide and (2) the 2xe2x80x2-O-methyl oligonucleotide containing duplex was not a substrate for ribonuclease H.
Dunlap et al., Biochemistry, 10: 2581-2587 (1971) evaluated degradation studies using 2xe2x80x2-O-methylated oligonucleotides with a crude cell-free protein synthesizing system known to contain a variety of nucleases and determined the methylated nucleotides conferred nuclease resistance to the polymers. The 2xe2x80x2-O-methylated oligonucleotides were very resistant to mixtures of alkaline phosphatase, snake venom phosphodiesterase, and micrococcal nuclease.
The present invention provides methods for increasing hammerhead ribozyme catalytic activity which methods comprise introducing into a flanking sequence of the hammerhead ribozyme nucleotide modified to have a substituent on the oxygen at the 2xe2x80x2 position, i.e., a 2xe2x80x2-O-substituted nucleotide.
The invention also provides hammerhead ribozymes comprising a flanking sequence nucleotide modified to have a substituent on the oxygen at the 2xe2x80x2 position.
In addition, the invention provides methods for increasing ribozyme catalytic activity comprising contacting an RNA molecule with a ribozyme having a flanking sequence nucleotide modified to have a substituent on the oxygen at the 2xe2x80x2 position, and a facilitator oligonucleotide.
The present invention further provides compositions comprising a ribozyme having modified flanking sequences and an effective amount of a facilitator oligonucleotide.
The present invention also provides ribozymes comprising a flanking sequence modified to contain 2xe2x80x2-O-alkyl or acyl nucleotides.